Endotoxin Analysis
Endotoxins are lipopolysaccharides (LPS) found in the outer membrane of the cell-wall of Gram-negative bacteria. Endotoxins comprise a lipid part, called lipid A, a core oligosaccharide making up the backbone of the macromolecule, and an O-antigen consisting of a variety of repeating oligosaccharide residues. It is lipid A that confers toxicity to the molecule.
Endotoxins are powerful pyrogens and cause i.a. fever, meningitis and a rapid fall in blood pressure if introduced into blood or tissues of the body. Components of the outer membrane such as endotoxins are released into the environment when Gram-negative bacteria lyse or divide, resulting in contamination of the environment. This contamination is difficult to prevent because endotoxins are ubiquitous, stable and small enough to pass through conventional sterilizing filters.
It is therefore of great importance to test pharmaceutical preparations and medical equipment that will be introduced into the body of a patient for endotoxin contamination. The method presently preferred to detect endotoxins is based on a lysate of amebocytes from the blood of the horseshoe crab, Limulus polyphemus. An alternative method is the rabbit pyrogenicity test, wherein a sample suspected of containing endotoxin is injected into a rabbit while monitoring the rabbit's body temperature.
The Limulus Amebocyte Lysate (LAL) method comprise four reaction steps, see FIG. 1. It is based on a cascade of enzyme activation steps terminating in the cleavage of the peptide coagulogen. This results in insoluble cleavage products, coagulin which coalesce by ionic interaction. If a sufficient amount of coagulin forms, turbidity appears followed by a gel-clot. The clotting enzyme that cleaves coagulogen also cleaves other peptides comprising a cleavage site similar to that in coagulogen. This has been used to construct peptides with such a cleavage site and a chromophore, paranitroanilide (pNA). Cleavage of this peptide results in the release of pNA which is yellow and absorbs light at 405 nm. The release of pNA can thus be measured in a chromogenic assay. The LAL method is further described in FDA guidelines (1987) and ANSI/AAMI standard ST72:2002.
A major disadvantage of the LAL method is that a number of substances interfere with the method in its different steps and care must be taken to keep such substances from interfering. Examples of such substances are heparin, yeast and mould cell wall material and cellulosic material.
Another disadvantage of the LAL method is that the components of the lysate degrade quickly and the lysate consequently has a limited shelf life. Producing the lysate also includes the drawing of blood from live crabs. About 10-15% of the crabs do not survive this treatment and it is estimated that 20 000-37 500 crabs die each year following this treatment. Furthermore, as any product isolated from nature, the exact composition of the lysate differs between batches, which affects the reproducibility of the method.
Chaby, R. reviewed a number of LPS-binding molecules in Cellular and Molecular Life Sciences, vol. 61 (2004) pp 1697-1713. The ongoing attempts to find endotoxin detection reagents were acknowledged by the author. But even though Chaby notes that the enzymatic activity of OmpT requires the ligation of LPS, it is not suggested by this author that this property of OmpT can be used in an assay for detection of endotoxin.
There is thus a great need for a quicker, cheaper, more reliable and animal friendly method for endotoxin analysis. There is also a need for more stable reagents for use in such a method.
Outer Membrane Protease T
The outer membrane protease T, OmpT, is a component of the outer membrane of E. coli. It has given name to the serine peptidase family S18, omptin. OmpT has been suggested to be involved in urinary tract disease since ompT genes were found in clinical isolates of E. coli. It has also been suggested that OmpT participates in the degradation of antimicrobial peptides secreted by epithelial cells from the urinary tract. However, the general biological function of OmpT remains to be elucidated. It has recently been found to be dependent on lipopolysaccharides for its activity (Kramer, R. A (2000), Brandenburg, K. et al (2005)). These publications do however not disclose that LPS may be detected in a sample by measuring the OmpT activity.